In this study, we investigated the mechanism of interleukin-4 (IL-4)protection against acetaminophen-induced liver injury in mice. Wild type (WT) and IL-4 deficient mice (IL-4 KO) were treated with an hepatotoxic dose of acetaminophen (APAP). IL-4 KO mice were found to be more susceptible to AILI. Their enhanced susceptibility was associated with prolonged depression of hepatic glutathione (GSH), which is a major cellular antioxidant and protective factor, that appeared to be due to reduced levels of gamma-glutamylcysteine ligase (gamma-GCL), the rate determining step in GSH synthesis. Other studies suggested that the low levels of gamma-GCL in the livers of IL-4 KO mice were not due to enhanced turnover of gamma-GCL, but instead caused by decreased gene expression as a result of upstream signaling pathways being depressed, including those mediated by the transcription factors, activating protein-1 (AP-1), and nuclear factor erythroid 2 related factor-2 (Nrf-2). Moreover, administration of IL-4 to IL-4 KO mice post-APAP treatment diminished the severity of liver injury and increased gamma-GCL and GSH levels. Recent studies suggest that IL-4 may be directly acting on hepatocytes as the IL-4 receptor has been detected on the surface of these cells. Conclusion: Overall these results are consistent with IL-4 having a previously unrecognized role in regulating the synthesis of GSH under conditions of cellular stress and as such having a non-redundant role in reducing susceptibility of mice and possibly humans to not only AILI, but also to liver injury caused by other drugs. Moreover, it possible that patients who are susceptible to DILD have polymorphisms in the IL-4 gene, the IL-4 receptor gene, and/or possibly the gamma-GCL gene that leads to low levels of hepatic GSH.